Chondrocytes in the growth plate progress through steps of resting, proliferation, maturation, and hypertrophy and finally become terminally differentiated cells that produce a mature mineralized matrix and support bone formation. As differentiation proceeds, chondrocytes express unique marker genes indicative of specific stages of differentiation. The cellular and molecular bases underlying the steps in growth plate differentiation remain largely unknown. This is largely due to a lack of information on the functional properties of chondrocytes at each different stage. A major obstacle in obtaining this information is the heterogeneous nature of growth plate chondrocytes. Density gradient centrifugation has been used to separate these chondrocytes into subpopulations, but it is unclear that differences in size and density accurately reflect stages of chondrocyte differentiation. This technique also requires starting with a large number of cells. This is not easily adapted to the mouse growth plate, and there are numerous transgenic and gene "knock-out" mice with striking growth plate abnormalities. It would be ideal to have a technique that could reliably separate out functionally distinct populations of chondrocytes from the mouse growth plate. We propose to develop a cell-sorting technique based on the hypothesis that differentiation stagespecific growth plate chondrocytes can be identified and isolated according to the promoter activities of the marker genes expressed at discrete stages of differentiation. We will also test the hypothesis that the signal transduction pathways that are active in chondrocytes change with the stage of differentiation. In Aim 1 we will express the promoter elements for the type II collagen (alpha1subunit, cyclin D1, type X collagen alpha1 subunit, and osteopontin genes, which are pre-tagged with distinct fluorescent protein cDNAs in chondrocytes using replication-deficient adenoviruses. We will sort by FACS different subpopulations of chondrocytes according to the level of activation of promoters, reflected in different fluorescent signal intensities. In Aim 2, we will determine whether the profile of signal transduction responses to parathyroid hormone related protein (PTHrP), an important modulator of chondrocyte differentiation, differs in chondrocytes at different stages of differentiation. The effects of PTHrP on cyclic AMP (cAMP) formation, phospholipase C (PLC) activity, and intracellular Ca 2. mobilization will be examined in different subpopulations of chondrocytes.